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Differential effect of electrical stimulation and β-adrenergic stimulation on neonatal rat ventricular myocyte monolayer conduction properties

S.P. Wells,1,2 H.M.M. Waddell,1 L.M.D. Delbridge1 and J.R. Bell,1 1Department of Physiology, University of Melbourne, VIC 3010, Australia and 2Institute of Cardiovascular Sciences, University of Birmingham, B15 2TT, United Kingdom.

Background: To develop novel treatments for cardiac arrhythmias, the electrical disturbances underpinning the arrhythmic phenotype in different settings requires more complete understanding. Multielectrode arrays (MEAs) provide a useful tool to non-invasively map cardiac electrophysiology in both cardiomyocyte monolayer cultures and atrial/ventricular tissue slices. Primary neonatal rat ventricular myocytes (NRVMs) represent an attractive model for studying inter-myocyte conduction properties in vitro, as cultures can be maintained beating spontaneously as a monolayer for 7+ days. Additionally, the capacity for short/long-term pharmacological treatment and/or gene manipulation (e.g. adenoviral infection) of NRVM cultures allows for investigation of the molecular mechanisms mediating inter-cardiomyocyte conduction. At present, there is limited characterisation data available regarding fundamental field potential and conduction properties in both spontaneously beating and electrically paced NRVMs.

Aim: The aim of this study was to characterize the basic electrophysiological properties of spontaneously beating and electrically-paced NRVM monolayers as a prelude to further studies assessing the molecular mechanisms of conduction heterogeneities.

Method: Hearts were removed from two-day old, anaesthetized (80% ethanol), neonatal Sprague-Dawley rats and cardiomyocytes isolated by collagenase and trypsin digestion. NRVMs (30,000 cardiomyocytes in 75 μl) were seeded onto the centre of fibronectin-coated (10 μg/ml) MEA chips (60PedotEcoMEA, MultiChannel Systems; electrode spacing 700 μm, diameter 100 μm, layout 8 × 8) and maintained in culture for 5-6 days. Field potentials were measured and conduction maps generated from spontaneously beating NRVMs at 37°C in the absence and presence of 1 μM isoproterenol using a filtered digital signal (high pass = 0.1 Hz, low pass = 3.5 kHz) with an MEA2100 system (MultiChannel Systems) sampling at 10 kHz. Paced electrical activity was recorded at 10% and 50% above the spontaneous beating rate using 2× capture threshold bipolar pulses (400 μs/phase) from the electrode nearest the spontaneous activity pacemaker. Results are presented as mean ± SEM. Comparisons between groups were performed with a Student’s paired t-test or one-way ANOVA, as appropriate. Differences were considered significant at P<0.05.

Results: NRVMs were responsive to β-adrenergic stimulation, with spontaneous beating rate significantly increased in the presence of isoproterenol (basal vs isoproterenol-treated: 86 ± 6 bpm vs 144 ± 17 bpm, n=3, P<0.05). This was associated with a small, but significant increase in conduction velocity (23.37 ± 1.82 cm/s vs 24.92 ± 2.06 cm/s, n=3, P<0.05) and no change in field potential amplitude (3.17 ± 0.25 mV vs 3.09 ± 0.24 mV, n=3, P=0.50). In contrast, conduction velocity was significantly decreased in NRVMs electrically paced at rates 10% and 50% greater than basal (spontaneous beating rate vs spontaneous beating rate +10% vs spontaneous beating rate +50%, 24.22 ± 1.32 cm/s vs 18.07 ± 0.34 cm/s vs 18.73 ± 0.75 cm/s, n=3, P<0.05). Field potential amplitude remained unchanged, consistent with isoproterenol-treated NRVMs.

Conclusions: These preliminary findings showing NRVM responses to isoproterenol treatment and electrical stimulation, provide evidence to support the use of NRVMs as a suitable model for assessing cardiac conduction properties in vitro. The positive chronotropic response to isoproterenol treatment was associated with an increased conduction velocity, whereas electrical pacing was associated with decreased conduction velocity. This observation would be consistent with a β-adrenergic mediated phosphorylation of gap junction proteins, a mechanism which requires confirmation in the NRVM setting. This study demonstrates a first step in the validation of NRVMs as a model for assessing conduction properties in spontaneously beating cardiomyocyte cultures. Further studies will be performed to interrogate mechanisms underlying changes in cardiomyocyte conduction linked with arrhythmia vulnerability.